The process of angiogenesis involves a complex interplay of biological functions including (1) "activation" of quiescent endothelium, (2) vascular endothelial cell invasion of basement membranes and adjacent tissues and (3) capillary tube formation. These events can be stimulated by a number of different angiogenic factors. Angiogenic factors can, however, have quite different effects on capillary endothelial cell locomotion and proliferation "in vitro," two of the key events necessary for the formation of new capillary blood vessels. Some angiogenic factors stimulate endothelial cell locomotion or proliferation, or both. In contrast, others have no effect, or inhibit endothelial cell proliferation "in vitro." These findings suggest that various angiogenic factors may operate either directly or indirectly when evaluated according to their putative targets.
Human basic fibroblast growth factor (bFGF) is classified as a "direct" angiogenic factor. A human bFGF molecule that was purified from placenta ("placental bFGF") was shown to (a) stimulate capillary endothelial cell proliferation, (b) to stimulate chemotaxis in capillary endothelial cells and (c) to stimulate these same cells to produce plasminogen activator and latent collagenase. Moscatelli et al., Proc. Natl. Acad. Sci. USA, 1986, Vol. 83, p. 2091. "In vivo," the plasminogen activator can convert the zymogen plasminogen to active plasmin, a protease of wide specificity. The plasmin can then convert latent collagenase to active collagenase. Thus, under the influence of the placental bFGF, capillary endothelial cells can generate two proteases that are able to degrade most of the proteins in surrounding tissues, which would allow the endothelial cells to penetrate the tissues. Indeed, the purified placental bFGF protein was shown to be angiogenic "in vivo." See Moscatelli supra; Squires et al., J. Bio. Chem., 1988, in press (est. December 1988 publication). The isolation, structure and properties of the human placental bFGF have been described in U.S. patent application Ser. No. 163,142 of Moscatelli et al., which is incorporated herein in its entirety by this reference.
An angiogenic protein in a pure form, such as the placental bFGF molecule just described, can be developed into a therapeutically valuable material. Because of the biological properties of the placental bFGF, the protein, when properly administered, can have beneficial effects in the healing of wounds and bone defects, in the repair of cardiovascular damage, repair of arteriosclerotic lesions and endothelialization of synthetic vascular grafts. In addition, the placental bFGF has been shown to have neurotrophic properties which might be beneficial in the treatment of neurological disorders of diverse origins.
Several proteins have been identified which have been referred to as "angiogenic factors." Many of these proteins were isolated from nonhuman sources. There is reason to believe that angiogenic factors isolated from nonhuman sources would not be suitable for use as therapeutic agents in humans due to the potential for adverse immunological reaction in response to a foreign protein.
The nucleotide sequence of a cDNA encoding the human bFGF protein was first published by Abraham. See Abraham et al., EMBO, 1986, Vol. 5, pp. 2523-28. Based on the position of a single putative initiator methionine codon in the cDNA, these authors predicted that the bFGF gene product would consist of a protein of 154 amino acids ("bFGF-18"). Sommer and coworkers have shown, however, that the bFGF preparation isolated from human placenta contained a bFGF species that was N-terminally extended relative to the gene product that was predicted by Abraham et al. from the bFGF cDNA. See Sommer et al., Biochem. Biophys. Res. Commun., 1987, Vol. 144, pp. 543-550.
These findings suggested the existence of multiple forms of human bFGF angiogenic factors that have not yet been described.
Based on this framework of research, the present inventors sought and discovered three new molecular forms of human bFGF classified here according to their approximate molecular size in kilodaltons (kD) as bFGF-22, bFGF-23 and bFGF-24. Collectively, the three new proteins will be referred to as higher molecular weight bFGFs (hmwbFGFs) in contrast to the previously characterized and in the literature described bFGF molecules of approximate molecular weight of 18 kD (bFGF-18) and the placental bFGF species which contains two additional amino acids N-terminal to bFGF-18 initiated methionine.
The hmwbFGF,s are substantially homologous to those isolatable from the human hepatoma cell line SK-HEP-1, and have at least one active site possessing an activity selected from the group consisting of mitogenic activity, chemotactic activity, angiogenic activity, the ability to stimulate protease synthesis, and combinations thereof.
In addition to discovering the hmwbFGF's, the present inventors have also discovered the first example of a normal animal cell gene initiating protein synthesis in vivo at a non-ATG codon. Although non-ATG initiation has been described in procaryotic cell genes, there is only one previous report of animal cell genes exhibiting this behavior. Hann et al., Cell, 1988, Vol. 52 pp. 185-189. Hann has reported, using in vitro translations, that the cMYC proto-oncogene also appears to utilize non-ATG codons for translation initiation.
The existence of higher molecular weight forms of bFGF in which translation was initiated at non-ATG codons suggests that similar higher molecular weight species may exist for other human proteins of therapeutic value. The higher molecular weight forms of the protein may have similar, enhanced or even new therapeutic qualities than the parent protein. Recognizing that translational initiation may also begin at a non-ATG codon prior to an identified putative (ATG) initiator will allow researchers to seek higher molecular weight forms of many proteins. Presumably, higher molecular weight forms of active proteins, if any, will possess similar therapeutic activities while also possessing unknown additional benefits or qualities. The additional amino-terminal peptide segments may be beneficial in altering or shutting off various active sites in the proteins, or in helping control the mobility of the protein or direct its location within or to the outside of the cell. The present invention includes high molecular weight forms of therapeutic proteins that are synthesized in vivo by translation initiation from non-ATG codons, in addition to the specific example of hmwbFGF's.
The preferred hmwbFGF angiogenic factors according to the present invention have the bFGF-18 core amino acid sequence shown as follows: ##STR1## In addition, peptides having the sequences L-G-G-R-G-R-G-R-A-P-E-R-V-G-G-R-G-R-G-R -G-T-A-A-P-R-A-A-P-A-A-R-G-S-R-P-G-P-A-G-T
L-P-G-G-R-L-G-G-R-G-R-G-R-A-P-E-R-V-G-G -R-G-R-G-R-G-T-A-A-P-R-A-A-P-A-A-R-G-S-R -P-G-P-A-G-T PA0 L-G-A-R-G-R-A-L-P-G-G-R-L-G-G-R-G-R-G-R -A-P-E-R-V-G-G-R-G-R-G-R-G-T-A-A-P-R-A-A -P-A-A-R-G-S-R-P-G-P-A-G-T PA0 L-G-G-R-G-R-G-R-A-P-E-R-V-G-G-R-G-R-G-R -G-T-A-A-P-R-A-A-P-A-A-R-G-S-R-P-G-P-A-G -T-bFGF-18 PA0 L-P-G-G-R-L-G-G-R-G-R-G-R-A-P-E-R-V-G-G -R-G-R-G-R-G-T-A-A-P-R-A-A-P-A-A-R-G-S-R -P-G-P-A-G-T-bFGF-18 PA0 L-G-A-R-G-R-A-L-P-G-G-R-L-G-G-R-G-R-G-R -A-P-E-R-V-G-G-R-G-R-G-R-G-T-A-A-P-R-A-A -P-A-A-R-G-S-R-P-G-P-A-G-T-bFGF-18
are present in the polypeptides outside the core sequence. Among the particularly preferred hmwbFGF angiogenic factors are the following sequences:
The amino acids represented by the foreqoing abbreviations are set forth in the description of the preferred embodiments below.
The relevant nucleotide sequence of the cDNA clone used to generate RNA for translation leading to the hmwbFGF's and the bFGF-18 factor is as follows: ##STR2## The bFGF-18 polypeptide is initiated at ATG 365, although, as described previously, the placental bFGF has a two-amino acid amino-terminal extension unto the methionine formed by the ATG initiator. The nucleotide numbering begins at the first nucleic acid of the cDNA clone that has been identified as expressing the bFGF angiogenic factor. The nucleotide sequence of the cDNA clone used to generate RNA for translation leading to the bFGF-18 factor is as follows:
__________________________________________________________________________ ATG GCA GCC GGG AGC ATC ACC ACG CTG CCC GCC TTG CCC GAG GAT GGC GGC AGC GGC GCC TTC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG TAC TGC AAA AAG GGG GGC TTC TTC CTG CGC ATC CAC CCC GAC GGC CGA GTT GAC GGG GTC CGG GAG AAG AGC GAC CCT CAC ATC AAG CTA CAA CTT CAA GCA GAA GAG AGA GGA bFGF-18 GTT GTG TCT ATC AAA GGA GTG TGT GCT AAC CGT TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT GTT ACG GAT GAG TGT TTC TTT TTT GAA CGA TTG GAA TCT AAT AAC TAC AAT ACT TAC CGG TCA AGG AAA TAC ACC AGT TGG TAT GTG GCA CTG AAA CGA ACT GGG CAG TAT AAA CTT GGA TCC AAA ACA GGA CCT GGG CAG AAA GCT ATA CTT TTT CTT CCA ATG TCT GCT AAG AGC TGA TTT TAA __________________________________________________________________________
The preferred hmwbFGF's are produced by translation initiation beginning at codons prior to ATG 365. The particularly preferred hmwbFGF's are produced by translation initiation at CTG 201, CTG 228 and CTG 243. The nucleic acids represented by the foregoing abbreviations are set forth in the Description of the Preferred Embodiments below.
Furthermore, in accordance with the present invention, pharmaceutical compositions containing, as at least one of the active ingredients, an angiogenic factor in accordance with the present invention as set forth herein are disclosed.
It is to be understood that both the foregoing general description and the following detailed descriptions are exemplary and exemplary only and are not restrictive of the invention, as claimed.